Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

Abstract

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

Structural transitions in Φ29 DNAP-DNA complexes critical to the translocation step and to dNTP binding. Shown are crystal structure models for the Φ29 DNAP-DNA, post-translocation state binary complex in the fingers-open conformation (Protein Data Bank entry 2PZS) (A) and the Φ29 DNAP-DNA-dNTP, post-translocation state ternary complex in the fingers-closed conformation (Protein Data Bank entry 2PYJ) (B). C and D, close-up views of the polymerase active site from the structures shown in A and B, respectively. The structures are from Ref. and were determined using the D12A/D66A mutant of Φ29 DNAP. In A and B, the protein backbone is rendered as a gray ribbon, with residues 359–395 in the fingers domain in red ribbon to highlight the conformation difference between the open binary complex and the closed ternary complex. The backbone positions of the Asp-12 and Asp-66 residues in the exonuclease domain are colored magenta. In A–D, the DNA primer strand is displayed in orange, the DNA template strand is yellow, and the templating base at n = 0 is in cyan. Residues Tyr-254, Tyr-226, and Tyr-390 are rendered in blue (space-filling in A and B, sticks in C and D). In B and D, the incoming dNTP is shown in green. In A and C, the side chains of Tyr-254 and Tyr-390 are stacked, obstructing the dNTP binding site; in B and D, both tyrosine side chains are rotated out of the stacking interaction, removing the steric impediment to the incoming dNTP. In C and D, the water molecule that mediates the interaction of the hydroxyl group of Tyr-390 with the −1 and −2 residues of the template strand of the duplex is shown as a red sphere. This water is part of an extensive network of water-mediated interactions with the minor groove of the active site-proximal duplex, a network that is precisely conserved between Φ29 DNAP and the B-family DNAP from bacteriophage RB69 (). The black dashed lines indicate potential hydrogen bonding interactions for the hydroxyl groups of the Tyr-226 or Tyr-390 side chains, including the hydrogen bond between the two side chains (labeled 2.7 Å in D). In C, the dashed gray line between the hydroxyl groups of the Tyr-226 and Tyr-390 side chains in the binary complex illustrates the increased distance between the hydroxyl groups of Tyr-226 and Tyr-390 (>5 Å) when the fingers are in the open conformation.

Capture of Φ29 DNAP-DNA complexes on the α-HL nanopore. In the nanopore device (A), a single α-HL nanopore is inserted in a ∼25-μm diameter lipid bilayer separating two chambers (cis and trans) that contain buffer solution. A patch clamp amplifier applies voltage across the bilayer and measures ionic current, which is carried through the nanopore by K+ and Cl− ions. B, DNA1 is a hairpin, featuring a 14-base pair duplex and a single-stranded template region of 35 nucleotides. The primer strand is terminated with a 2′-H, 3′-H CMP residue, and the template strand contains a reporter group of five consecutive abasic (1′-H, 2′-H) residues spanning positions +8 to +12 (indicated as red letters X in the sequence). C, representative current trace for a binary complex formed between Φ29 DNAP and the DNA1 substrate, captured at 180 mV applied potential in buffer containing 10 mm K-Hepes, pH 8.0, 0.3 m KCl, 1 mm EDTA, 1 mm DTT, and 11 mm MgCl2. DNA and Φ29 DNAP were added to the nanopore cis chamber to final concentrations of 1 and 0.75 μm, respectively. Schematics diagrams above the current trace illustrate the sequence of events, which is described in the Introduction. In the schematic diagrams, the five consecutive abasic (1′, 2′-H) residues spanning positions +8 to +12 of the template strand, which serve as a reporter group, are shown as red circles. D, ionic current traces for Φ29 DNAP = DNA1 complexes, captured at 180 mV in the presence of 0 μm (i) or 40 μm (ii) dGTP. E, a three-state model in which translocation and dNTP binding are sequential: dNTP can bind to complexes (kon [dNTP]) only after the transition from the pre-translocation to the post-translocation state (r1); the transition from the post-translocation to the pre-translocation state (r2) cannot occur before the dissociation of dNTP (koff).

Processive DNA synthesis catalyzed by the Y226F mutant.A, DNA synthesis catalyzed by the wild type Φ29 DNAP (lanes 1–6) or the Y226F mutant (lanes 7–12) as a function of enzyme concentration. A series of 2-fold serial dilutions of each enzyme was tested, in which the highest concentration (lanes 1 and 7, indicated by enzyme dilution = 1) was 30 pm; the reactions were conducted at 30 °C for 30 min. B, DNA synthesis as a function of time for reactions catalyzed by the wild type Φ29 DNAP (lanes 1–5) or the Y226F mutant (lanes 6–10). Reactions were conducted using an enzyme concentration of 60 pm at 30 °C for the indicated times. In both A and B, the replication substrate was oligonucleotide-primed bacteriophage M13 single-stranded DNA (∼3.35 pm). The reaction products were resolved by electrophoresis in alkaline agarose gels.

Transition rates of the Φ29 DNAP translocation step extracted from ionic current traces measured in absence of dNTP. Shown are plots of log(r1) versus voltage (A) and log(r2) versus voltage (B) for binary complexes formed between the wild type (blue squares), Y226F (red squares), Y390F (yellow squares), D12A/D66A (blue triangles), Y226F/D12A/D66A (red triangles), or Y390F/D12A/D66A (yellow triangles) Φ29 DNAP and DNA1. Each plotted point shows the mean ± S.E. In the absence of dNTP, the fluctuation rates between the pre-translocation and post-translocation states are fully described by a two-state model with two transition rates (). Each plotted data point shows the means ± S.E., determined from 15–30 ionic current time traces for individual captured complexes; each time trace had a duration of 5–10 s.

Complementary dNTP binding affinities of Φ29 DNAP mutants. The normalized p/(1 − p) (where p is the probability of post-translocation state occupancy, and the normalized p/(1 − p), defined as the value of p/(1 − p) in the presence of a given concentration of dNTP, divided by the value for p/(1 − p) for the same Φ29 DNAP-DNA complex at 0 μm dGTP ()) is plotted (A) as a function of dGTP concentration for complexes formed between wild type, Y226F, and Y390F Φ29 DNAP. In B, (normalized p/(1 − p)) − 1 is plotted on a log scale as a function of dGTP concentration for complexes formed between the wild type, Y226F, Y390F, D12A/D66A, Y226F/D12A/D66A, or Y390F/D12A/D66A Φ29 DNAP and DNA1. Complexes were captured at 180 mV. Plot symbols for each of the enzymes are given in the legend to . Error bars, S.E. Each data point was determined from 15–30 ionic current time traces for individual captured complexes; each time trace had a duration of 5–10 s.

Translocation rates and dNTP association and dissociation rates determined simultaneously from ionic current traces measured in the presence of dNTP. Shown are plots of log(r1) versus voltage (A) and log(r2) versus voltage (B) for complexes formed between the wild type, Y226F, Y390F, D12A/D66A, Y226F/D12A/D66A, and Y390F/D12A/D66A Φ29 DNAP and DNA1, captured in the presence of dGTP. Also shown are plots of konversus voltage (C) and koffversus voltage (D) for complexes formed between the wild type, Y226F, Y390F, D12A/D66A, Y226F/D12A/D66A, and Y390F/D12A/D66A Φ29 DNAP and DNA1, captured in the presence of dGTP. Rates were extracted from ionic current traces using the autocorrelation function and the three-state model shown in E. Plot symbols for each of the enzymes are given in the legend to . Error bars, S.E. Each data point was determined from 15–30 ionic current time traces for individual captured complexes; each time trace had a duration of 5–10 s. The data plotted are for complexes captured in the presence of the following dGTP concentrations: wild type, 10 μm; D12A/D66A, 10 μm; Y226F, 5 μm; Y390F, 20 μm; Y226F/D12A/D66A, 5 μm; and Y390F/D12A/D66A, 20 μm. Although we have shown that all four transition rates (r1, r2, kon, and koff) are independent of [dNTP] (), the method of extracting the rates from the ionic current traces using autocorrelation and the three-state model (E) is most robust when using data collected under conditions where all three states are well sampled. For example, when the dNTP concentration is very low, the dGTP-bound state is not well sampled; when the dNTP concentration is very high, only the dGTP-bound state is well sampled. The dNTP concentrations optimal for the analysis vary with the dNTP binding affinity of each mutant.

Effects of Mn2+ on dNTP binding to mutant Φ29 DNAP-DNA complexes.A, the (normalized p/(1 − p)) − 1 is plotted on a log scale as a function of dGTP concentration for complexes formed between DNA1 and the D12A/D66A mutant in the presence of Mg2+ (blue triangles) or Mn2+ (dark green triangles) and between DNA1 and the Y390F/D12A/D66A mutant in the presence of Mg2+ (yellow triangles) or Mn2+ (light green triangles). Complexes were captured at 180 mV. The data are plotted according to the concentration of dGTP added to the nanopore cis chamber. Because a fraction of the added dGTP is bound by complexes in the bulk phase (∼100-μl cis chamber volume, in which [DNA1] = 1 μm, and [enzyme] = 0.75 μm), the free [dGTP] is lower than the input [dGTP]. The difference is significant when the input [dGTP] is comparable with or lower than the concentration of complexes. This accounts for the difference between the data for D12A/D66A mutant in the presence of Mn2+ (A, dark green triangles) and the linear fitting because the linear relation is with respect to the free [dGTP]. Shown are plots of r1versus [dGTP] (B) and r2versus [dGTP] (C) for complexes formed between the D12A/D66A mutant or the Y390F/D12A/D66A mutant and DNA1, captured at 180 mV in the presence of Mg2+ or Mn2+. Because there is no zero value on the log scale plot, in B and C, the values of r1 and r2 for binary complexes of the two mutants, captured in the presence of Mg2+ or Mn2+ are placed on the plot at the position for 0.2 μm dGTP and are indicated by an arrow and label (0 μm dGTP). The binary complex translocation rates were determined using autocorrelation and the two-state model (); the translocation rates in the presence of dGTP were determined using autocorrelation and the three-state model (E) (). Shown are plots of konversus [dGTP] (D) and koffversus [dGTP] (E) for complexes formed between the D12A/D66A mutant or the Y390F/D12A/D66A mutant and DNA1, captured at 180 mV in the presence of Mg2+ or Mn2+. The dNTP binding rates were determined using autocorrelation and the three-state model. Plot symbols in B–D are the same as in A. Each data point was determined from 15–30 ionic current time traces for individual captured complexes; each time trace had a duration of 5–10 s. Error bars, S.E.

Pyrophosphate binding to mutant Φ29 DNAP-DNA complexes.A, the normalized (1 − p)/p (where (1 − p) is the probability of pre-translocation state occupancy, and the normalized (1 − p)/p is the value of (1 − p)/p in the presence of a given concentration of pyrophosphate, divided by the value for (1 − p)/p for the same Φ29 DNAP-DNA complex at 0 mm pyrophosphate) is plotted as a function of pyrophosphate concentration for complexes formed between wild type, Y226F, Y390F, D12A/D66A, Y226F/D12A/D66A, or Y390F/D12A/D66A Φ29 DNAP and DNA1. Plot symbols for each of the enzymes are given in the legend to . In B, the mean dwell times in the pre-translocation and post-translocation states for complexes formed between wild type Φ29 DNAP (blue squares, pre-translocation; blue circles, post-translocation) and the Y226F mutant (red squares, pre-translocation; red stars, post-translocation) are plotted as a function of pyrophosphate concentration. Complexes were captured at 160 mV. Each data point was determined from 15–30 ionic current time traces for individual captured complexes; each time trace had a duration of 5–10 s. Error bars, S.E.